generation of functional biomarkers in prostate cancer · generation of function based biomarkers...
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Generation of Function Generation of Function Based Biomarkers in Based Biomarkers in
Prostate CancerProstate CancerK.C. Balaji, MDK.C. Balaji, MD
Wake Forest University School of Medicine,Wake Forest University School of Medicine,Winston Salem, NCWinston Salem, NC
Substance used as an indicator of a Substance used as an indicator of a biological statebiological state
Can be used to monitor a normal physiologic Can be used to monitor a normal physiologic state, a pathologic process, a pharmacologic state, a pathologic process, a pharmacologic response to therapeutic intervention, or a response to therapeutic intervention, or a toxic exposuretoxic exposure
Examples: antibodies, radioactive isotopes, Examples: antibodies, radioactive isotopes, specific DNA sequences, specific DNA sequences, PSAPSA
Phases of Biomarker DevelopmentPhases of Biomarker Development
Fig. 1 PSA clinical course and biomarker uses.
Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3Published by AAAS
Prostate Specific Antigen (PSA) as Prostate Specific Antigen (PSA) as a Biomarker in Prostate Cancera Biomarker in Prostate Cancer
United States Cancer statistics, 2012United States Cancer statistics, 2012
CA: A Cancer Journal for Clinicians
Volume 62, Issue 1, pages 10-29, 4 JAN 2012 DOI: 10.3322/caac.20138 http://onlinelibrary.wiley.com/doi/10.3322/caac.20138/full#fig1
Cumulative Hazard of Death from Prostate Cancer among Men 55 to 69 Years of Age.
Schröder
FH et al. N Engl
J Med 2012;366:981-990.
Number of Diagnoses of All Prostate Cancers (Panel A) and Number
of Prostate-Cancer Deaths (Panel B).
Andriole
GL et al. N Engl
J Med 2009;360:1310-
1319.
Why is PSA Screening for Why is PSA Screening for Prostate Cancer Prostate Cancer Controversial?Controversial?
For startersFor starters………….PSA is NOT a .PSA is NOT a rationale based biomarkerrationale based biomarker
Prostate Specific Antigen (hK3)Prostate Specific Antigen (hK3)PSA made by prostate tissue PSA made by prostate tissue –– benign and benign and malignant tissuemalignant tissueSink effect Sink effect –– performance to monitor malignant performance to monitor malignant progression improves when prostate tissue is progression improves when prostate tissue is minimalminimalSerine protease of the human Serine protease of the human kallikreinkallikrein familyfamilyLiquefies the coagulum by acting on Liquefies the coagulum by acting on semenogellinsemenogellin I & II and FibronectinI & II and FibronectinProduced as a precursor moleculeProduced as a precursor molecule-- the prothe pro--PSAPSA
HYPOTHESISHYPOTHESIS
Function based biomarkers Function based biomarkers may demonstrate improved may demonstrate improved clinical performance clinical performance characteristicscharacteristics
Cancer PhenotypeCancer Phenotype
Benign Cells
Cancer Cells
Proliferation
Migration
Invasion
Cancer Biology Cancer Biology --
Hallmarks of CancerHallmarks of Cancer
Hanahan
D, Weinberg RA. 2011
Future challenges and unmet needs in prostate cancer biomarker research.
Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3
Published by AAAS
OPPORTUNITIES IN PROSTATE OPPORTUNITIES IN PROSTATE CANCERCANCER
Metastatic CascadeMetastatic Cascade
Castration Resistant Prostate Castration Resistant Prostate Cancer (CRPC)Cancer (CRPC)
Schulman et al., European Urology, Vol 58, 1, pages e1 – e18, July 2010
Differential gene expressionDifferential gene expression The modelThe model
Derived from human Derived from human prostate cancer prostate cancer lymph nodal lymph nodal metastasismetastasisAndrogen Androgen dependentdependentProduce PSAProduce PSA
Derived from LNCaP Derived from LNCaP cells grown in presence cells grown in presence of bone stromal cellsof bone stromal cellsLow steady state of Low steady state of androgen receptorandrogen receptorAndrogen independentAndrogen independentHighly metastatic and Highly metastatic and produces osteoblastic produces osteoblastic lesions / produce PSAlesions / produce PSA
LNCaP cells C4-2 cells
Wu et al., Int J Ca, 57, 406-12, 1994
Differential gene expressionDifferential gene expression Experimental designExperimental design
LNCaP tRNA C4-2 tRNA
mRNA mRNA
MICROARRAY
1: RPA Data
2: Western Blotting
3: Kinase activity
4: PKD1 -
down regulated in advanced human prostate cancer
LNCaP C4-2
Protein Kinase D1 (PKD1) is Down Regulated in Protein Kinase D1 (PKD1) is Down Regulated in Advanced Prostate CancerAdvanced Prostate Cancer
PKD1 is downregulated in breast cancer Eiseler et al. Breast Cancer Res, 2009 and in gastric cancer Kim et al. Carcinogenesis. 2008
Varambally et al. Cancer Cell 2005
Phosphatidylserine, Ca2+, Diacylglycerol/phorbol ester,
DAG + PKC
ACTIVATORS:- Regulatory peptides (neurotensin, bombesin)- Lysophosphatidic acid, - Thrombin that act through Gq, G12, Gi, and Rho, - Growth factors, such as PDGF and IGF- B/T cell antigen engagement,- Oxidative stress
Tissues and Cells: - Fibroblasts - Intestinal and kidney epithelial cells - Smooth muscle cells- Cardiac myocytes- Neuronal cells - Osteoblasts- B and T lymphocytes,- Mast cells and platelets, - Several human cancers
Protein Kinase D1 (PKD1)Protein Kinase D1 (PKD1)
LNCaP cells were stained with antibody specific for PKD1 (A) is DIC image (B) is showing immunolocalization of PKD1. (C) is merge of image (A) and (B) showing perinuclear and junctional staining ( Jaggi et al., 2003).
Localization of PKD1 in LNCaP cells7
A B
C
DIC PKD1
MERGE
Molecular Biology of the Cell 3rd Edition, Figure 19-18
tight junction
adherens
junction
desmosome
gap junction
hemi-desmosome
microvilli
keratin filaments
basal lamina
band of actin filaments
Epithelial cellsEpithelial cells
Cadherin
β-catenin/plakoglobin
α-catenin
Actin filaments
ZO-1
p120ctn
vinculinα-actinin
Cadherin–Catenin Protein Complex
Wheelock et al. Current Topics in Membranes (1996)
PKD1 interacts and phosphorylates E-cadherinLNCaP
PKD-1 and E-Cadherin regulate PC cells proliferation and soft agar colony formation
PKD-1 and E-Cadherin regulate PC cells proliferation and soft agar colony formation –
Corroboration with molecular markers
β-Catenin mediates the PKD1 and E-cadherin functions in PC cells
Cadherin
β-catenin/plakoglobin
α-catenin
Actin filaments
ZO-1
p120ctn
vinculinα-actinin
Cadherin–Catenin Protein Complex
Wheelock et al. Current Topics in Membranes (1996)
PKD1 promotes β-catenin membrane trafficking
PKD1 is associated with membrane trafficking of β-catenin
Totalβ−catenine(α HA tag)
pT120
3T3
WT
T102
I/T
112R
/T12
0I
T120
I
pS916 PKD1
pT120 β-catenin
total β-catenin
total PKD1
- + Bryostatin
C4-2/PKD1
C4-
2C
4-2/
PKD
1C
4-2
C4-
2/PK
D1
+ + + pT120-
-
+ normal peptide-
+ -
phosphopeptide
C4-2/PKD1
H102 pT120
autoradiographStaining
active PKD1 - + -dead PKD1 - - +
Raamfp etldegmqipsTqfdaahpT nvqR
PKD1 phosphorylates β-catenin at Thr120
β-cat H102 Ab
p230
β-cat pT120 Ab
p230
β-catenin
α-catenin
Active BetaActive Beta--catenin catenin (unphosphorylated S37/T41) in the Nucleus(unphosphorylated S37/T41) in the Nucleus
Maher et al., PLoS One. 2010; 5(4): e10184
PKD1 represses generation of ABCPKD1 represses generation of ABC
Table 1. Analysis of pT120 antibody staining in prostate cancer
TransGolgi
network staining
Two-sample t test (P value)
n positive (%) negative (%)
Normal vs. tumor Gleason 3-6 vs. 7-10
Normal
22 16 (72.7%)
6 (27.3%)
Total tumor 200 15 (7.5%) 185 (92.5%)
P<0.01
Gleason3-6
27 4 (14.8%) 23 (85.2%)7-10
173 11 (6.3%) 162 (93.7%)
P<0.05
Table 2. Comparison of H102 and pT120 staining patterns
Total
H102 staining
pT120 staining
Normal tissue
membranous β-catenin 9
8 (88.9%)TGN β-catenin
9
8 (88.9%)
Tumor tissue
increased
expression
56
18 (32.1%)
9 (16.1%)decreased expression 56
5 (8.9%)
5 (8.9%)membranous
54
42 (77.8%)
8 (14.3%)cytoplasma/nuclear
54
12 (22.2%)
6 (10.7%)
EE--cadherin and Betacadherin and Beta--catenin Expression are catenin Expression are Down Regulated in High Risk Prostate Cancer Down Regulated in High Risk Prostate Cancer
(6F9 monoclonal anti(6F9 monoclonal anti--beta catenin COOH terminal antibody)beta catenin COOH terminal antibody)
E-cadh Beta-cat
PIN
Nuc-Beta Cat
TGN staining of pT120 BetaTGN staining of pT120 Beta-- catenin antibodycatenin antibody
OPPORTUNITIESOPPORTUNITIESPhosphoPhospho--specific Betaspecific Beta--catenin characterize catenin characterize functional changes in cellsfunctional changes in cellsUnderstanding postUnderstanding post--translational modifications translational modifications and implications on biological functions can and implications on biological functions can facilitate development of rationale based facilitate development of rationale based biomarkersbiomarkersHYPOTHESISHYPOTHESIS: Generation of function based : Generation of function based biomarkers will improve performance biomarkers will improve performance characteristics of biomarkers in clinical settingcharacteristics of biomarkers in clinical setting
AcknowledgementsAcknowledgements Mentors, Staff and ColleaguesMentors, Staff and Colleagues
University of Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NEOmaha, NEUniversity of Massachusetts Medical University of Massachusetts Medical School, Worcester, MASchool, Worcester, MAWake Forest University School of Wake Forest University School of Medicine, Winston Salem, NCMedicine, Winston Salem, NC
Funding: DoD, VA, Prostate Cancer Foundation –
Univ
of Neb Med Ctr, UMass Med Sch, Wake Forest Univ
Sch
of Med Cancer Center, WFU Institute of Regen
Med
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